Nuclear-to-cytoplasmic ratios of 1PN and 2PN zygotes after in vitro fertilization of mouse oocytes.

Numerous studies have reported comparisons of the nuclear-to-cytoplasmic (NC) ratio during mitosis. However, little information is known about how the pronuclear size is regulated and determined at the end of meiosis II in mammalian zygotes. The present study aims to analyze the NC ratio of female and male pronuclei, and also to compare the size of single pronuclei using photographs that were obtained during experiments to create chimeric hermaphrodites from 2-cell oocytes. The volume of both the female and the male pronucleus was found to correlate with the volume of the oocyte cytoplasm. The NC ratio of the male pronucleus was greater than that of the female pronucleus. The NC ratio of the average volume of the female and male pronuclei was greater than the NC ratio of the mononucleate oocytes. The occurrence of 1PN oocytes was significantly higher when the volume of cytoplasm was lower than the cut-off value. These results indicated that the NC ratio is retained during pronuclear formation. A higher NC ratio in male compared with the female pronucleus indicated structural and/or molecular difference between the two pronuclei. 1PN formation may occur when sperm enters close to the MII spindle.

Aggregated chromosomes transfer in human oocytes.

Germinal-vesicle (GV) transfer, spindle-chromosome complex transfer in metaphase-II oocytes and two pronuclei transfer have been evaluated as possible treatments for patients who have mitochondrial diseases. However, GV transfers often lead to heteroplasmy while the other two methods are frequently associated with aneuploidy. The present study used a new method based on the transfer of aggregated chromosomes, which occurs in human oocytes, before the metaphase spindle is established.

Symmetrical division of mouse oocytes during meiotic maturation can lead to the development of twin embryos that amalgamate to form a chimeric hermaphrodite.

BACKGROUND: Gentle compression of mouse oocytes during meiosis-1 prevented the usual extrusion of a small polar body and resulted in the symmetrical division of the ooplasm into two cells of similar size within the zona pellucida. The purpose of our study was to determine whether such cells, equivalent to two small oocytes, were capable of embryonic development and would result in birth following transfer to the uterus. METHODS: IVF of the 2-celled oocytes was performed and the twin intra-zonal embryos were observed. In each case, the two embryos that originated from fertilized cells with two pronuclei were observed to amalgamate and form a single morula and subsequent blastocyst that was transferred to the uterus of a recipient of a different mouse strain. FISH analysis was performed on sectioned paraffin-embedded tissue of the offspring. RESULTS: In symmetrically divided oocytes each cell contained a metaphase II spindle. Both cells were fertilizable and cleaved to form twin embryos within the same zona pellucida. Most twin embryos amalgamated to form a single compacted morula, which progressed to hatched blastocysts that contained a single inner cell mass. In total, 104 of these blastocysts were transferred to 19 mice, two of which became pregnant, resulting in the birth of three offspring. FISH analysis showed that one newborn contained both XX and XY cells. CONCLUSIONS: We found that two small oocytes fertilized within the same zona pellucida to form twin embryos that amalgamate to establish a single chimeric embryo. This may be one mechanism that leads to the formation of a chimeric hermaphrodite when an embryo containing XX cells mixes with its intra-zonal twin containing XY cells.

Risk factors for term small for gestational age infants in women with low prepregnancy body mass index.

AIM: The purpose of our study was to investigate the association between low maternal prepregnancy body mass index (BMI) less than 18.5 kg/m(2) and the incidence of small for gestational age (SGA) infants. MATERIAL & METHODS: This was a cross-sectional study. The women with BMI of less than 25.0 kg/m(2) who gave birth to single term infants (37-42 weeks) at clinics and hospitals in the Tokyo metropolitan area between 2003 and 2004 were analyzed for risk factors for SGA. RESULTS: Five hundred and seventy-two women were underweight (BMI < 18.5 kg/m(2)) and 2708 (75.1%) were normal (18.5

Association of spindle midzone particles with polo-like kinase 1 during meiosis in mouse and human oocytes.

Polo-like kinase 1 (Plk1) has been reported to localize to the spindle midzone during meiosis in mouse oocytes. However, it has not been reported in human oocytes. In this study, the interaction of the meiotic structures and chromosome segregation in mouse and human oocytes were studied by time-lapse differential interference contrast microscopy. Using immunocytochemical studies, the localization of polo-like kinase 1 and its association with microtubules were examined during the extrusion of first and second polar bodies. It was found that Plk1 was localized in the spindle midzone in human oocytes at anaphase I and telophase I. Also, three-dimensional confocal laser microscopy showed that the meiotic spindle midzone contained numerous dot-like particles that were stained by anti-Plk1 antibody. These particles were aligned in the plane of the meiotic midzone in mouse and human oocytes.

A General Framework for Tilings, Delone Sets, Functions, and Measures and Their Interrelation.

We define a general framework that includes objects such as tilings, Delone sets, functions, and measures. We define local derivability and mutual local derivability (MLD) between any two of these objects in order to describe their interrelation. This is a generalization of the local derivability and MLD (or S-MLD) for tilings and Delone sets which are used in literature, under a mild assumption. We show that several canonical maps in aperiodic order send an object P to one that is MLD with P . Moreover, we show that, for an object P and a class Σ of objects, a mild condition on them ensures that there exists some Q ∈ Σ that is MLD with P . As an application, we study pattern-equivariant functions. In particular, we show that the space of all pattern-equivariant functions contains all the information on the original object up to MLD, in a quite general setting.

Dietary folate intake during pregnancy and birth weight in Japan.

BACKGROUND: Inadequate folate status has been associated with many negative reproductive outcomes, such as neural tube defects (NTD), low birth weight and placental abruption. AIM OF THE STUDY: The objectives of this study were to evaluate the levels of dietary folate intake during pregnancy in Japanese women and the subsequent birth weight of their babies. METHODS: A longitudinal prospective study was conducted with 197 women with a singleton pregnancy in 2005. Dietary folate was investigated 3 times: in the first trimester at 12 weeks, in the second trimester at 20 weeks and in third trimester at 32 weeks using a diet history questionnaire (DHQ). Non fasting blood samples were collected from the women for measurement of homocysteine, hemoglobin, ferritin, unbound iron-binding capacity (UIBC) and total iron-binding capacity (TIBC). RESULTS: Energy intake increased as pregnancy advanced, but not significantly. The daily intake of folate increased from 248.5 +/- 113.1 microg/d in the first trimester to 275.4 +/- 100.2 microg/d in the third trimester (P = 0.04). This was well below the recommended level of 440 microg/d and only 10% of mothers were above the levels. In the third trimester, plasma homocysteine concentration was significantly higher in the low folate group of less than 250 microg/d (P = 0.02), but not the first and second trimesters. Dietary folate intake and plasma homocysteine concentrations were not likely to be predictors of birth weight in our subjects. CONCLUSIONS: Our study shows that Japanese women's energy and folate intakes do not meet their energy needs during pregnancy and are at an extremely low recommended dietary allowance level throughout pregnancy.

Experience of sexual violence among sexually experienced Japanese teenage girls and influencing factors.

Aim: The aim of the present study was to identify influencing factors for sexual violence in sexually experienced teenage girls. Methods: Eighty sexually experienced teenage girls aged between 14 and 19 attending obstetrics and gynecology clinics in suburban Tokyo were selected as subjects. After completing a simple questionnaire on sexual behavior and lifestyle the subjects were interviewed using a semistructured interview technique. Logistic regression analysis was used to identify factors associated with sexual violence. Results: Police and government statistics for rape, sexual and domestic violence in Japan are extremely low. However, the present study showed that 52.5% of subjects had been victims of sexual violence. Furthermore, of these, 69.0% had been forced to have sex with a partner and 64.3% had been raped by someone other than a partner. Factors influencing the risk of sexual violence were sexual behavior and attitudes towards sexuality and lifestyle. Having a sexually transmitted infection was not an influencing factor. Conclusions: It is important to identify teenage girls attending obstetrics and gynecology clinics at risk of sexual violence and to teach them about the risks of sexual behavior associated with imbalances of power. Furthermore, it is essential to support victims of sexual violence to protect them from the recurrence of such abuse. (Reprod Med Biol 2008; 7: 181-187).

Restricting weight gain during pregnancy in Japan: a controversial factor in reducing perinatal complications.

OBJECTIVE: To evaluate the effectiveness of restricting weight gain during pregnancy to reduce perinatal complications. STUDY DESIGN: The study was conducted in the Tokyo metropolitan area, and reviewed 3071 mothers and their infants born from singleton pregnancies retrospectively. To examine the influence of increased maternal weight gain on perinatal complications, we performed five-category stratification for weight gain: less than 8.0, 8.0-10.0, 10.1-12.0, 12.1-14.0 and over 14.0 kg. RESULTS: Total weight gains less than 8.0 kg significantly increased the risk of low birth weight (LBW) and small for gestational age (SGA) infants (OR=2.19, 95% CI; 1.36-3.52, OR=1.76, 95% CI; 1.23-2.51) and total weight gain over 14.0 kg significantly increased the risk of large for gestational age (LGA) infants and pregnancy induced hypertension (PIH) (OR=3.06, 95% CI; 1.88-4.98, OR=2.87, 95% CI; 1.86-4.42, respectively), compared with women with weight gain of 10.1-12.0 kg. The groups with weight gains of 8.0-10.0 kg and 12.1-14.0 kg did not show adverse perinatal outcomes, including gestational diabetes (GDM), cesarean delivery, postpartum hemorrhage and laceration, significantly different from the 10.1 to 12.0 kg gain group. CONCLUSION: Strict restriction of weight gain during pregnancy is not effective in reducing perinatal complications.

Detection and quantification of male-specific fetal DNA in the serum of pregnant cynomolgus monkeys (Macaca fascicularis).

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175-131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.

The redox state of recombinant human serum albumin and its optimal concentration for mouse embryo culture.

Albumin has multiple physiological functions in embryo culture, such as a chelator of heavy metals, free radical scavenger, pH and osmotic regulator, a stabilizer, growth factor carrier, a surfactant, and a nutrient. However, the commercially available human serum albumin (HSA) products may not be completely safe since they could be contaminated with viruses and prions. Recombinant human serum albumin (rHSA) has been reported to be as efficient as commercial HSA for fertilization and embryo development. Despite the possible benefits of rHSA, it has not been widely used for embryo culture due to its high cost of production. Our objective was to analyze the redox state of different types of HSA products and rHSA to define oxidative status batch variations of HSA and rHSA and to evaluate the optimal concentration of rHSA for mouse embryo culture. The redox state of the HSA and rHSA used in embryo culture media was found to vary widely. Redox variations were found among different HSA batches as well as among rHSA batches. The highest blastocyst development and hatching rates were obtained with rHSA used at a concentration of 0.05 mg/mL. We showed that very low concentrations of rHSA were most favorable for advanced mouse embryo development in culture.

The influence of the redox state of follicular fluid albumin on the viability of aspirated human oocytes.

A number of reports have suggested that the oxidative state of human albumin in serum and in some body fluids is associated with cell damage. However there are no reports on the redox state of human follicular fluid (FF) and its influence on oocyte viability. The aim of this study was to examine the relationship between the redox state of FF and serum on oocyte viability. The cytoplasmic condition of oocytes was evaluated microscopically at collection in 117 women. Deteriorating oocytes were recognized by degenerative changes in their cytoplasm. The redox state of FFs that yielded degenerated oocytes was evaluated and compared with fluids containing normal oocytes. The redox state of the corresponding FF and serum, at the time of oocyte retrieval, was analyzed by high performance liquid chromatography. The redox state of FF that contained degenerated oocytes was found to have a significantly elevated oxidized state compared with the FFs that yielded normal oocytes. Also the albumin in the FF of patients was found to be predominantly in the reduced state compared with that in their serum at the time of oocyte retrieval. In addition, increasing age and endometriosis were found to shift the redox of serum to the oxidative state. We propose that the reduced state of albumin in FF may play an important role in protecting oocytes from oxidative damage.

Impact of dietary folate intake on depressive symptoms in young women of reproductive age.

INTRODUCTION: Folate deficiencies may be linked to depressive disorders among persons suffering from neurological and psychiatric problems. The purpose of the present study was to evaluate the impact of dietary folate intake on depressive symptoms in young Japanese women of reproductive age. METHODS: The study was conducted in Japan in 2009 with 141 Japanese women aged 18 to 28 years. A Japanese version of the Center for Epidemiologic Studies Depression (CES-D) scale was used to screen for depressive symptoms. Dietary intake was investigated by a diet history questionnaire. Nonfasting blood samples were collected from the women to measure folate and homocysteine levels. RESULTS: The proportion of women with lower folate intake (< 240 µg/d) was significantly higher in the women with CES-D scores greater than or equal to 19 when compared with the folate levels in those with CES-D scores less than 19 (75.0% vs 43.6%, respectively; P < .001). Logistic regression analysis revealed significant associations between CES-D scores greater than or equal to 19, low folate intake, and low vitamin B(6) intake when adjusted for age, living status, smoking, and body mass index. Adequate folate intake of more than the recommended dietary allowance (RDA) level of 240 µg per day was independently related to a decreased risk of depression (adjusted odds ratio 0.22; 95% confidence interval, 0.11-0.56; P < .001). DISCUSSION: Multiple logistic regression analysis revealed a reduced incidence of depression in women whose intake of folate exceeded the RDA of 240 µg per day. This finding suggests that dietary folate intake may be causally related to depressive symptoms in women of reproductive age. If results of studies powered to determine causal relationships are similarly positive, folate supplementation could reduce the incidence of depression.

Damage of embryo development caused by peroxidized mineral oil and its association with albumin in culture.

OBJECTIVES: To examine the effect of free radicals from peroxidized oil and the role of albumin on the passage of radicals. DESIGN: Prospective study. SETTING: Clinical IVF laboratory and university department. PATIENT(S): Blood samples were donated by laboratory staff. INTERVENTION(S): Examination of the effects of mineral oil samples with various peroxide value (POV) on culture of erythrocytes and on the passage of a lipophilic tracer, DiI, into the zona pellucida. MAIN OUTCOME MEASURE(S): Time required for hemolysis of red blood cells by peroxidized oil, staining of zona pellucida from human oocytes and embryos by lipophilic tracer, and POV analysis of mineral oil samples in relation to various storage conditions. RESULT(S): The time for hemolysis was related to the POV levels of oil samples covering the culture medium. Albumin in the medium facilitated hemolysis and the passage of DiI into the zona. Peroxidized oil (POV >0.02 meq/kg) blocked the entry of DiI into the zona. CONCLUSION(S): The presence of albumin in the medium was associated with the entry into the human zona of agents present in peroxidized mineral oil. This process and variable oil peroxidation could be deleterious to embryos in culture.

Dietary folate intake, use of folate supplements, lifestyle factors, and serum folate levels among pregnant women in Tokyo, Japan.

AIM: The aim of this study was to determine the folate status of pregnant women and how it is affected by dietary folate, use of folate supplements, and lifestyle factors (smoking and drinking habits). METHODS: This cross-sectional study involved 537 healthy, pregnant women attending an obstetric clinic in a Tokyo suburb. Morning blood samples were taken to determine serum folate levels. A brief, self-administered diet history questionnaire and another questionnaire were used to obtain information about food intake and demographic data. Logistic regression was used to identify factors associated with a normal serum folate level (> 6 ng/mL), taking women with a low serum folate level (< or = 6 ng/mL) as the reference group. RESULTS: The median dietary folate intake was 207.2 microg/day; the median serum folate level was 3.9 ng/ml. A normal serum folate level was significantly associated with increased dietary folate intake (odds ratio = 1.003, P < 0.05), use of folate supplements (odds ratio = 8.152, P < 0.001), and more than four cups of green tea (odds ratio = 0.467, P < 0.05), but not with lifestyle factors in the adjusted analysis. CONCLUSIONS: Most pregnant women (79.3%) in the present study had a low serum folate level, reflecting a low dietary intake of folate and a low proportion of women taking folate supplements. Supplement use had a much stronger association with a normal serum folate level than dietary folate intake. Since this study was not population-based, further studies are needed to confirm the results.

A phase of chromosome aggregation during meiosis in human oocytes.

A higher rate of chromosomal abnormality occurs in human oocytes compared with other animal oocytes. In this study, chromosome movement has been successfully observed during first and second meiosis using a time-lapse culture system and a differential interference contrast inverted microscope. In human oocytes, a specific sequence of early maturation changes was observed. Following the completion of nucleolar breakdown, chromosomes were assembled into a single aggregation that heralded the start of nuclear membrane breakdown. The chromosome aggregation phase (gere phase) persisted after germinal vesicle (GV) breakdown, lasting several hours, and a similar gere phase (chromosome gathering) occurred after the first polar body extrusion, lasting 1-4 h. In contrast, in mouse GV oocytes, nucleolar and nuclear membranes started to break down almost at the same time. A chromosome aggregation phase was not observed in mouse oocytes. The discovery of a gere phase during human oocyte maturation may provide important information related to the mechanism of abnormal chromosomal segregation, which often occurs during meiosis.

Lipofuscin bodies in human oocytes as an indicator of oocyte quality.

Refractile bodies are one of the main morphological abnormalities that can be observed in the cytoplasm of human oocytes. In the present studies the characteristics of refractile bodies and the relationship between the size of these structures and developmental competence of the affected oocytes and resulting embryos were examined. The refractile bodies were found to have yellow autofluorescence which was consistent with the typical autofluorescence of lipofuscin. Viewed by transmitted electron microscopy, the refractile bodies showed the conventional morphology of lipofuscin inclusions and consisted of a mixture of lipids and dense granule materials. Large refractile bodies (>5 microm) were positively stained by the Schmorl reaction and were considered to contain lipofuscin. These larger lipofuscin inclusions (>5 microm) were associated with significantly reduced fertilization and unfavorable blastocyst development.

Peroxidation of mineral oil used in droplet culture is detrimental to fertilization and embryo development.

Unexpected deterioration of human embryos led us to an investigation of possible toxic factors in the oil covering culture medium. Elevated levels of peroxidation in the oil by oxygen exposure or unfavorable storage were found to be the main causes of toxicity.

Risk factors for small for gestational age.

BACKGROUND: The purpose of the paper was to determine the risk factors for small-for-gestational-age (SGA) infants at full term, in Japan. METHODS: The study was conducted at four hospitals and clinics in the Tokyo metropolitan area. A retrospective review of 2972 mothers and their infants born from singleton pregnancies at any time during the years 2002 and 2003 was conducted. RESULTS: Of these women, 8.4% gave birth to SGA infants. The proportion of SGA infants was significantly higher among heavy smokers (>10 cigarettes/day; 13.7%, P < 0.01). The odds ratio (OR) for SGA decreased significantly in proportion to the pregnancy body mass index (OR, 0.89; 95% confidence interval [CI]: 0.84-0.94, P < 0.001). The OR of SGA for stratified maternal weight gain was 1.79 (95%CI: 1.24-2.58, P 12 kg. CONCLUSION: The present study clearly confirms the detrimental effect of a low prepregnancy body mass index, low maternal weight gain and maternal smoking during pregnancy on the incidence of SGA infants.

Timed IVM followed by ICSI in a patient with immature ovarian oocytes.

A complete failure of meiotic maturation occasionally occurs following human chorionic gonadotrophin administration during IVF-intracytoplasmic sperm injection (ICSI) cycles. ICSI on day 1 is commonly used to allow maturation in culture. However, if the oocytes become mature in the evening soon after their recovery but ICSI is delayed until the next day, then subsequent ageing of matured oocytes may be unfavourable for fertilization and development. To avoid the deterioration associated with oocyte ageing, the timing of polar body extrusion was checked every 3 h and rescue in-vitro maturation (IVM)-ICSI was performed shortly after the polar body extrusion was confirmed. This report describes a successful pregnancy and birth of a healthy baby in a patient who had no mature oocytes at the time of oocyte retrieval, and illustrates the value of extra monitoring for IVM and ICSI in cases where only immature oocytes are available.